1. Field of the Invention
The invention relates to the field of blocking peptides which interact with ligand growth factor receptors. Specifically, the blocking peptides of the invention are capable of binding to ligands to the epidermal growth factor receptor and the erbB-2 receptor. The present invention further relates to the use of such blocking peptides in assays to detect the presence of ligand growth factors. Methods of using the blocking peptides of the invention as a means of inhibiting the growth of adenocarcinoma cells and for diagnosing cancer are also disclosed.
2. Review of Related Art
Transforming growth factor ligands belong to a family of heat and acid-stable polypeptides which allow cells to assume a transformed morphology and form progressively growing colonies in anchorage-independent growth assays (DeLarco, et al., Proc. Natl. Acad. Sci. USA, 75:4001-4005 (1978); Moses, et al., Cancer Res., 41:2842-2848 (1981); Ozanne, et al., J. Cell. Physiol., 105:163-180 (1980); Roberts, et al., Proc. Natl. Acad. Sci. USA, 77:3494-3498 (1980)). The epidermal growth factor receptor (EGFR) and its physiologic ligands, epidermal growth factor (EGF) and transforming growth factor alpha (TGF.alpha.), play a prominent role in the growth regulation of many normal and malignant cell types (Carpenter, G., Annu. Rev. Biochem., 5:881-914 (1987)).
One role the EGF receptor system may play in the oncogenic growth of cells is through autocrine-stimulated growth. If cells express the EGFR and secrete EGF and/or TGF.alpha. then such cells could stimulate their own growth. Since some human breast cancer cell lines and tumors express EGFR (Osborne, et al., J. Clin. Endo. Metab., 55:86-93 (1982); Fitzpatrick, et al., Cancer Res., 44:3442-3447 (1984); Filmus, et al., Biochem. Biophys. Res. Commun., 128:898-905 (1985); Davidson, et al., Mol. Endocrinol., 1:216-223 (1987); Sainsbury, et al., Lancet, i: 1398-1402 (1987); Perez, et al., Cancer Res. Treat., 4:189-193 (1984)) and secrete TGF.alpha. A (Bates, et al., Cancer Res., 46:1707-1713 (1986); Bates, et al., Mol. Endocrinol., 2:543-555 (1988)), an autocrine growth stimulatory pathway has been proposed in breast cancer (Lippman, et al., Breast Cancer Res. Treat., 7:59-70 (1986)).
An autocrine growth stimulatory pathway analogous to that proposed for epidermal growth factor receptor and its ligands may also be employed by a growing list of oncogene encoded transmembrane proteins that have structure reminiscent of growth factor receptors. This list includes the protooncogenes neu and its human equivalent erbB-2 or HER2 (Bargmann, et al., Nature, 319:226-229 (1986); Coussens, et al., Science, 230:1131-1139 (1985); Yamamoto, et al., Nature, 319:230-234 (1986); c-kit (Yarden, et al., EMBO J., 6:341-3351 (1987); ros (Neckameyer, et al., Mol Cell. Biol., 6:1478-1486 (1986); met (Park, et al., Proc. Natl. Acad. Sci. USA, 84:6379-6383 (1987); trk (Martin-Zanca, et al., Nature, 319:743-748 (1986); and ret (Takahashi, et al., Mol. Cell. Biol., 7:1378-1385 (1987)). The erbB-2 and c-kit protooncogenes encode factors that display structural homology with EGFR (Yarden, et al., Annu. Rev. Biochem., 57:443-478 (1988). Although erbB-2 and its related oncogene neu are related to EGFR, these proteins are distinct. For example, known EGFR ligands such as EGF and TGF.alpha. do not bind to erbB-2 receptor. (King, et al., EMBO J., 7:1647 (1988); and Stern, et al., EMBO J., 7:995 (1988).
Amplification or overexpression or both have been described for the c-erbB-2 gene in a number of human adenomas (Semba, et al., Proc. Natl. Acad. Sci. USA, 82:6497 (1985); Van de Vijver, et al., Mol. Cell. Biol., 7:2019 (1987); Yokota, et al., Oncogene, 2:283 (1988); Slamon,et al., Science 244:707 (1989); Aasland, et al., Br. J. Cancer, 57:358 (1991); Cline, et al., Cancer, 60:2669 (1991)). Overexpression statistically correlates with short term relapse in breast (Slamon, et al., Science, 235:177 (1987); Walker, et al., Br. J. Cancer, 60:426 (1991); Wright, et at., Cancer Res., 49:2087 (1991)), ovarian (Berchuck, et al., Cancer Res. 50:4087 (1990)) and gastric cancers (Yonomura, et al., ibid., 51:1034 (1991)). Moreover, overexpression of p185.sup.erbB-2, the product of the erbB-2 oncogene, has been associated with tumor cell resistance to several cytotoxic mechanisms including actions of tumor necrosis factor on NIH 3T3, breast or ovarian cancer cells (Hudziak, et al., Proc. Natl. Acad. Sci. USA 85:5102 (1988); Lichtenstein, et al., Cancer Res., 50:7364 (1990)), and those of natural killer cells (Wiltshle, et al., Proc. Am. Assoc. Cancer. Res., 32:202 (1991)) or tamoxifen (Benz, et al., ibid.:211) in breast cancer cells. Therefore, p185.sup.erbB-2 oncoprotein may have a central role both in the development and progression of human tumors, and constitutes a preferential therapeutic target in neoplasia when overexpressed and clinically associated with poor prognosis.
On the basis of its structural similarity with the extracellular domain (ECD) of the EGFR, the p185.sup.erbB-2 ECD was thought to function in growth factor binding. Two candidate ligands for p185.sup.erbB-2 have been so far purified: (i) a 30 kDa glycoprotein (gp30), secreted by MDA-MB-231 human breast cancer cells, that binds and phosphorylates both EGFR and p185.sup.erbB-2 (Lupu, et al., Science, 249:1552 (1991) and U.S. application Ser. No. 07/528,438, filed May 25, 1990 abandoned), and (ii) a 75 kDa protein (p75), secreted from SK-Br-3 human breast cancer cells, that specifically binds and phosphorylates p185.sup.erbB-2 (Lupo, et al., Proc. Natl. Acad. Sci. USA, 89:2287 (1992) and U.S. patent application Ser. No. 07/640,497, filed Jan. 14, 1991 abandoned). The further potency of these two proteins in acting on the proliferation of p185.sup.erbB-2 overexpressing human breast cancer cells point out the role of gp30 and p75 as growth factors in mammary neoplasia. Since EGF and TFG.alpha. do not bind p185.sup.erbB-2 (Schecter, et al., Science, 229:976 (1985); King, et al., EMBO J., 7:164 (1988); and Stem, et al., EMBO J., 7:995 (1988)), p185.sup.erbB-2 and EGFR ECDs appear to interact with both common and specific ligands. Thus, as with the EGFR and its ligands, the ligands for erbB-2 receptor are involved in the growth regulation of malignant cells.
According to the autocrine growth stimulatory pathway, malignant cells are capable of secreting a potent tumor growth factor in vivo. Thus, the growth factor ligand may be detected in body fluids, much like human chorionic gonadotropin or .alpha.-fetoprotein, and could be used as a tumor marker and a prognostic variable. Studies suggest that TGF.alpha. activity can be detected in body fluids of cancer patients and that its presence may provide important information concerning the biology of a patient's tumor (Stromberg, et al., J. Cell. Biochem., 32:247-259 (1986); Twardzick, et al., J. Natl. Cancer Inst., 69:793-798 (1982); Sherwin, et al., Cancer Res., 43:403-407 (1983)).